Investigation of cooperativity in the binding of ligands to the D-2 dopamine receptor

The D 2 dopamine receptor exists as dimers or as higher-order oligomers, as determined from data from physical experiments. In this study, we sought evidence that this oligomerization leads to cooperativity by examining the binding of three radioligands ([H-3] nemonapride, [H-3] raclopride, and [H-3] spiperone) to D 2 dopamine receptors expressed in membranes of Sf9 cells. In saturation binding experiments, the three radioligands exhibited different B-max values, and the B-max values could be altered by the addition of sodium ions to assays. Despite labeling different numbers of sites, the different ligands were able to achieve full inhibition in competition experiments. Some ligand pairs also exhibited complex inhibition curves in these experiments. In radioligand dissociation experiments, the rate of dissociation of [H-3] nemonapride or [H-3] spiperone depended on the sodium ion concentration but was independent of the competing ligand. Although some of the data in this study are consistent with the behavior of a cooperative oligomeric receptor, not all of the data are in agreement with this model. It may, therefore, be necessary to consider more complex models for the behavior of this receptor.

Large-scale functional expression of WT and truncated human adenosine A(2A) receptor in Pichia pastoris bioreactor cultures

Background: The large-scale production of G-protein coupled receptors (GPCRs) for functional and structural studies remains a challenge. Recent successes have been made in the expression of a range of GPCRs using Pichia pastoris as an expression host. P. pastoris has a number of advantages over other expression systems including ability to post-translationally modify expressed proteins, relative low cost for production and ability to grow to very high cell densities. Several previous studies have described the expression of GPCRs in P. pastoris using shaker flasks, which allow culturing of small volumes (500 ml) with moderate cell densities (OD600 similar to 15). The use of bioreactors, which allow straightforward culturing of large volumes, together with optimal control of growth parameters including pH and dissolved oxygen to maximise cell densities and expression of the target receptors, are an attractive alternative. The aim of this study was to compare the levels of expression of the human Adenosine 2A receptor (A(2A)R) in P. pastoris under control of a methanol-inducible promoter in both flask and bioreactor cultures. Results: Bioreactor cultures yielded an approximately five times increase in cell density (OD600 similar to 75) compared to flask cultures prior to induction and a doubling in functional expression level per mg of membrane protein, representing a significant optimisation. Furthermore, analysis of a C-terminally truncated A2AR, terminating at residue V334 yielded the highest levels (200 pmol/mg) so far reported for expression of this receptor in P. pastoris. This truncated form of the receptor was also revealed to be resistant to C-terminal degradation in contrast to the WT A(2A)R, and therefore more suitable for further functional and structural studies. Conclusion: Large-scale expression of the A(2A)R in P. pastoris bioreactor cultures results in significant increases in functional expression compared to traditional flask cultures.

An RNA molecule that specifically inhibits G-protein-coupled receptor kinase 2 in vitro

G-protein-coupled receptors are desensitized by a two-step process. In a first step, G-protein-coupled receptor kinases (GRKs) phosphorylate agonist-activated receptors that subsequently bind to a second class of proteins, the arrestins. GRKs can be classified into three subfamilies, which have been implicated in various diseases. The physiological role(s) of GRKs have been difficult to study as selective inhibitors are not available. We have used SELEX (systematic evolution of ligands by exponential enrichment) to develop RNA aptamers that potently and selectively inhibit GRK2. This process has yielded an aptamer, C13, which bound to GRK2 with a high affinity and inhibited GRK2-catalyzed rhodopsin phosphorylation with an IC50 of 4.1 nM. Phosphorylation of rhodopsin catalyzed by GRK5 was also inhibited, albeit with 20-fold lower potency (IC50 of 79 nM). Furthermore, C13 reveals significant specificity, since almost no inhibitory activity was detectable testing it against a panel of 14 other kinases. The aptamer is two orders of magnitude more potent than the best GRK2 inhibitors described previously and shows high selectivity for the GRK family of protein kinases.

Variation in pituitary expression of mRNAs encoding the putative inhibin co-receptor (betaglycan) and type-I and type-II activin receptors during the chicken ovulatory cycle

Secretion of LH and FSH from the anterior pituitary is regulated primarily by hypothalamic GnRH and ovarian steroid hormones. More recent evidence indicates regulatory roles for certain members of the transforming growth factor beta (TGF beta) superfamily including inhibin and activin. The aim of this study was to identify expression of mRNAs encoding key receptors and ligands of the inhibin/activin system in the hen pituitary gland and to monitor their expression throughout the 24-25-h ovulatory cycle. Hens maintained on long days (16 h light/8 h dark) were killed 20, 12, 6 and 2 h before predicted ovulation of a midsequence egg (n = 8 per group). Anterior pituitary glands were removed, RNA extracted and cDNA synthesized. Plasma concentrations of LH, FSH, progesterone and inhibin A were measured. Real-time quantitative PCR was used to quantify pituitary expression of mRNAs encoding betaglycan, activin receptor (ActR) subtypes (type I, IIA), GnRH receptor (GnP,H-R), LH beta subunit, FSH beta subunit and GAPDH. Levels of mRNA for inhibin/activin beta A and beta B subunits, inhibin alpha subunit, follistatin and ActRIIB mRNA in pituitary were undetectable by quantitative PCR (< 2 amol/reaction). Significant changes in expression (P < 0.05) of ActRIIA and betaglycan mRNA were found, both peaking 6 h before ovulation just prior to the preovulatory LH surge and reaching a nadir 2 h before ovulation, just after the LH surge. There were no significant changes in expression of ActRI mRNA throughout the cycle although values were correlated with mRNA levels for both ActRIIA (r=0.77; P < 0.001) and betaglycan (r=0.45; P < 0.01). Expression of GnRH-R mRNA was lowest 20 h before ovulation and highest (P < 0.05) 6 h before ovulation; values were weakly correlated with betaglycan (r=0.33; P=0.06) and ActRIIA (r=0.34; P=0.06) mRNA levels. Expression of mRNAs encoding LH beta and FSH beta subunit were both lowest (P < 0.05) after the LH surge, 2 h before ovulation. These results are consistent with an endocrine, but not a local intrapituitary, role of inhibin-related proteins in modulating gonadotroph function during the ovulatory cycle of the hen, potentially through interaction with betaglycan and ActRIIA. In contrast to mammals, intrapituitary expression of inhibin/activin subunits and follistatin appears to be extremely low or absent in the domestic fowl.

beta-arrestin binding to the beta(2)-adrenergic receptor requires both receptor phosphorylation and receptor activation

Homologous desensitization of beta(2)-adrenergic receptors has been shown to be mediated by phosphorylation of the agonist-stimulated receptor by G-protein-coupled receptor kinase 2 (GRK2) followed by binding of beta-arrestins to the phosphorylated receptor. Binding of beta-arrestin to the receptor is a prerequisite for subsequent receptor desensitization, internalization via clathrin-coated pits, and the initiation of alternative signaling pathways. In this study we have investigated the interactions between receptors and beta-arrestin2 in living cells using fluorescence resonance energy transfer. We show that (a) the initial kinetics of beta-arrestin2 binding to the receptor is limited by the kinetics of GRK2-mediated receptor phosphorylation; (b) repeated stimulation leads to the accumulation of GRK2-phosphorylated receptor, which can bind beta-arrestin2 very rapidly; and (c) the interaction of beta-arrestin2 with the receptor depends on the activation of the receptor by agonist because agonist withdrawal leads to swift dissociation of the receptor-beta-arrestin2 complex. This fast agonist-controlled association and dissociation of beta-arrestins from prephosphorylated receptors should permit rapid control of receptor sensitivity in repeatedly stimulated cells such as neurons.

Agonist-selective, receptor-specific interaction of human P-2Y receptors with beta-arrestin-1 and-2

Interaction of G-protein-coupled receptors with beta-arrestins is an important step in receptor desensitization and in triggering "alternative" signals. By means of confocal microscopy and fluorescence resonance energy transfer, we have investigated the internalization of the human P2Y receptors 1, 2, 4, 6, 11, and 12 and their interaction with beta-arrestin-1 and -2. Co-transfection of each individual P2Y receptor with beta-arrestin-1-GFP or beta-arrestin-2-YFP into HEK-293 cells and stimulation with the corresponding agonists resulted in a receptor-specific interaction pattern. The P2Y(1) receptor stimulated with ADP strongly translocated beta-arrestin-2-YFP, whereas only a slight translocation was observed for beta-arrestin-1-GFP. The P2Y(4) receptor exhibited equally strong translocation for beta-arrestin-1-GFP and beta-arrestin-2YFP when stimulated with UTP. The P2Y(6), P2Y(11), and P2Y(12) receptor internalized only when GRK2 was additionally cotransfected, but beta-arrestin translocation was only visible for the P2Y(6) and P2Y(11) receptor. The P2Y(2) receptor showed a beta-arrestin translocation pattern that was dependent on the agonist used for stimulation. UTP translocated beta-arrestin-1-GFP and beta-arrestin-2-YFP equally well, whereas ATP translocated beta-arrestin-1-GFP to a much lower extent than beta-arrestin2- YFP. The same agonist-dependent pattern was seen in fluorescence resonance energy transfer experiments between the fluorescently labeled P2Y(2) receptor and beta-arrestins. Thus, the P2Y(2) receptor would be classified as a class A receptor when stimulated with ATP or as a class B receptor when stimulated with UTP. The ligand-specific recruitment of beta-arrestins by ATP and UTP stimulation of P2Y(2) receptors was further found to result in differential stimulation of ERK phosphorylation. This suggests that the two different agonists induce distinct active states of this receptor that show differential interactions with beta-arrestins.

Allosteric effects of antagonists on signalling by the chemokine receptor CCR5

Antagonists of the chemokine receptor, CCRS, may provide important new drugs for the treatment of HIV-1. In this study we have examined the mechanism of action of two functional antagonists of the chemokine receptor CCRS (UK-396,794, UK-438,235) in signalling and internalisation assays using CHO cells expressing CCR5. Both compounds were potent inverse agonists versus agonist-independent [S-3]GTP gamma S binding to membranes of CHO cells expressing CCR5. Both compounds also acted as allosteric inhibitors of CCL5 (RANTES) and CCL8 (MCP-2) -stimulated [S-35]GTP gamma S binding to CHO-CCR5 membranes, reducing the potency and maximal effects of the two chemokines. The data are consistent with effects of the allosteric inhibitors on both the binding and signalling of the chemokines. Both compounds inhibited CCR5 internalisation triggered by chemokines. When CHO-CCR5 cells were treated with either of the two compounds for prolonged periods of time (24 h) an increase (similar to 15%) in cell surface CCRS was detected. (C) 2007 Elsevier Inc. All rights reserved

Inhibition of 125I-epidermal growth factor binding to murine fibroblasts and keratinocytes by irradiation with ultraviolet-B light

Treatment of murine Swiss 3T3 fibroblasts and XB/2 keratinocytes with UV-B light (302 nm) resulted in a dose-dependent inhibition of [125I] epidermal growth factor (EGF) binding. The light dose required to achieve 50% inhibition of binding in both cell types was 80–85 J/m2 Decreased [125I] platelet-derived growth factor binding was not evoked even by light doses of up to 280 J/m2 UV-B irradiation did not stimultate phosphorylation of the 80 kd protein substrate for protein kinase C. Furthermore, its effect on [125I]EGF binding was not altered as a consequence of protein kinase C down-regulation following prolonged exposure of cells to phorbol esters. These results indicate that UV-B-induced transmodulation of the epidermal growth factor receptor is a specific event mediated through a protein kinase C-indepen dent pathway. Transfer of culture medium from irradiated cells to untreated control cells showed this effect was not induced as a result of transforming growth factor α release and subsequent binding to the EGF receptor in these cells.

Leptin receptor polymorphisms interact with polyunsaturated fatty acids to augment risk of insulin resistance and metabolic syndrome in adults.

The leptin receptor (LEPR) is associated with insulin resistance, a key feature of metabolic syndrome (MetS). Gene-fatty acid interactions may affect MetS risk. The objective was to investigate the relationship among LEPR polymorphisms, insulin resistance, and MetS risk and whether plasma fatty acids, a biomarker of dietary fatty acids, modulate this. LEPR polymorphisms (rs10493380, rs1137100, rs1137101, rs12067936, rs1805096, rs2025805, rs3790419, rs3790433, rs6673324, and rs8179183), biochemical measurements, and plasma fatty acid profiles were determined in the LIPGENE-SU.VI.MAX study of MetS cases and matched controls (n = 1754). LEPR rs3790433 GG homozygotes had increased MetS risk compared with the minor A allele carriers [odds ratio (OR) = 1.65; 95% CI: 1.05–2.57; P = 0.028], which may be accounted for by their increased risk of elevated insulin concentrations (OR 2.40; 95% CI: 1.28–4.50; P = 0.006) and insulin resistance (OR = 2.15; 95% CI: 1.18–3.90; P = 0.012). Low (less than median) plasma (n-3) and high (n-6) PUFA status exacerbated the genetic risk conferred by GG homozygosity to hyperinsulinemia (OR 2.92–2.94) and insulin resistance (OR 3.40–3.47). Interestingly, these associations were abolished against a high (n-3) or low (n-6) PUFA background. Importantly, we replicated some of these findings in an independent cohort. Homozygosity for the LEPR rs3790433 G allele was associated with insulin resistance, which may predispose to increased MetS risk. Novel gene-nutrient interactions between LEPR rs3790433 and PUFA suggest that these genetic influences were more evident in individuals with low plasma (n-3) or high plasma (n-6) PUFA.

Modulation of interleukin signalling and gene expression in cardiac myocytes by endothelin-1

The related inflammatory cytokines, interleukin- (IL-) 1β and IL-33, are both implicated in the response of the heart to injury. They also activate mitogen-activated protein kinases (MAPKs) in cardiac myocytes. The hypertrophic Gq protein-coupled receptor agonist endothelin-1 is a potentially cardioprotective peptide and may modulate the inflammatory response. Endothelin-1 also stimulates (MAPKs) in cardiac myocytes and promotes rapid changes in expression of mRNAs encoding intercellular and intracellular signalling components including receptors for IL-33 (ST2) and phosphoprotein phosphatases. Prior exposure to endothelin-1 may specifically modulate the response to IL-33 and, more globally, influence MAPK activation by different stimuli. Neonatal rat ventricular myocytes were exposed to IL-1β or IL-33 with or without pre-exposure to endothelin-1 (5 h) and MAPK activation assessed. IL-33 activated ERK1/2, JNKs and p38-MAPK, but to a lesser degree than IL-1β. Endothelin-1 increased expression of soluble IL-33 receptors (sST2 receptors) which may prevent binding of IL-33 to the cell-surface receptors. However, pretreatment with endothelin-1 only inhibited activation of p38-MAPK by IL-33 with no significant influence on ERK1/2 and a small increase in activation of JNKs. Inhibition of p38-MAPK signalling following pretreatment with endothelin-1 was also detected with IL-1β, H2O2 or tumour necrosis factor α (TNFα) indicating an effect intrinsic to the signalling pathway. Endothelin-1 pretreatment suppressed the increase in expression of IL-6 mRNA induced by IL-1β and decreased the duration of expression of TNFα mRNA. Coupled with the general decrease in p38-MAPK signalling, we conclude that endothelin-1 attenuates the cardiac myocyte inflammatory response, potentially to confer cardioprotection.

Effects of the Allosteric Antagonist 1-(4-Chlorophenyl)-3-[3-(6-pyrrolidin-1-ylpyridin-2-yl)phenyl]urea (PSNCBAM-1) on CB1 Receptor Modulation in the Cerebellum

PSNCBAM-1 has recently been described as a cannabinoid CB1 receptor allosteric antagonist associated with hypophagic effects in vivo; however, PSNCBAM-1 effects on CB1 ligand-mediated modulation of neuronal excitability remain unknown. Here, we investigate PSNCBAM-1 actions on CB1 receptor-stimulated [35S]GTPγS binding in cerebellar membranes and on CB1 ligand modulation of presynaptic CB1 receptors at inhibitory interneurone-Purkinje cell (IN-PC) synapses in the cerebellum using whole-cell electrophysiology. PSNCBAM-1 caused non-competitive antagonism in [35S]GTPγS binding studies, with higher potency against the CB receptor agonist CP55940 than for WIN55,212-2 (WIN55). In electrophysiological studies, WIN55 and CP55940 reduced miniature inhibitory postsynaptic currents (mIPSCs) frequency, but not amplitude. PSNCBAM-1 application alone had no effect on mIPSCs; however, PSNCBAM-1 pre-treatment revealed agonist-dependent functional antagonism, abolishing CP55940-induced reductions in mIPSC frequency, but having no clear effect on WIN55 actions. The CB1 antagonist/inverse agonist AM251 increased mIPSC frequency beyond control, this effect was reversed by PSNCBAM-1. PSNCBAM-1 pre-treatment also attenuated AM251 effects. Thus, PSNCBAM-1 reduced CB1 receptor ligand functional efficacy in the cerebellum. The differential effect of PSNCBAM-1 on CP55940 versus WIN55 actions in [35S]GTPγS binding and electrophysiological studies and the attenuation of AM251 effects are consistent with the ligand-dependency associated with allosteric modulation. These data provide the first description of functional PSNCBAM-1 allosteric antagonist effects on neuronal excitability in the mammalian CNS. PSNCBAM-1 allosteric antagonism may provide viable therapeutic alternatives to orthosteric CB1 antagonists/inverse agonists in the treatment of CNS disease.

Variation in the human Cannabinoid Receptor (CNR1) gene modulates gaze duration for happy faces

BACKGROUND: Humans from an early age look longer at preferred stimuli, and also typically look longer at facial expressions of emotion, particularly happy faces. Atypical gaze patterns towards social stimuli are common in Autism Spectrum Conditions (ASC). However, it is unknown if gaze fixation patterns have any genetic basis. In this study, we tested if variations in the cannabinoid receptor 1 (CNR1) gene are associated with gaze duration towards happy faces. This gene was selected because CNR1 is a key component of the endocannabinoid system, involved in processing reward, and in our previous fMRI study we found variations in CNR1 modulates the striatal response to happy (but not disgust) faces. The striatum is involved in guiding gaze to rewarding aspects of a visual scene. We aimed to validate and extend this result in another sample using a different technique (gaze tracking). METHODS: 30 volunteers (13 males, 17 females) from the general population observed dynamic emotion expressions on a screen while their eye movements were recorded. They were genotyped for the identical four SNPs in the CNR1 gene tested in our earlier fMRI study. RESULTS: Two SNPs (rs806377 and rs806380) were associated with differential gaze duration for happy (but not disgust) faces. Importantly, the allelic groups associated with greater striatal response to happy faces in the fMRI study were associated with longer gaze duration for happy faces. CONCLUSIONS: These results suggest CNR1 variations modulate striatal function that underlies the perception of signals of social reward such as happy faces. This suggests CNR1 is a key element in the molecular architecture of perception of certain basic emotions. This may have implications for understanding neurodevelopmental conditions marked by atypical eye contact and facial emotion processing, such as ASC.

Analysis of protein networks in resting and collagen receptor (GPVI)-stimulated platelet sub-proteomes.

Proteomics approaches have made important contributions to the characterisation of platelet regulatory mechanisms. A common problem encountered with this method, however, is the masking of low-abundance (e.g. signalling) proteins in complex mixtures by highly abundant proteins. In this study, subcellular fractionation of washed human platelets either inactivated or stimulated with the glycoprotein (GP) VI collagen receptor agonist, collagen-related peptide, reduced the complexity of the platelet proteome. The majority of proteins identified by tandem mass spectrometry are involved in signalling. The effect of GPVI stimulation on levels of specific proteins in subcellular compartments was compared and analysed using in silico quantification, and protein associations were predicted using STRING (the search tool for recurring instances of neighbouring genes/proteins). Interestingly, we observed that some proteins that were previously unidentified in platelets including teneurin-1 and Van Gogh-like protein 1, translocated to the membrane upon GPVI stimulation. Newly identified proteins may be involved in GPVI signalling nodes of importance for haemostasis and thrombosis.

Feedback regulation by Atf3 in the endothelin-1-responsive transcriptome of cardiomyocytes: Egr1 is a principal Atf3 target

Endothelin-1 promotes cardiomyocyte hypertrophy by inducing changes in gene expression. Immediate early genes including activating transcription factor 3 (Atf3), Egr1 and Ptgs2 are rapidly and transiently upregulated by endothelin-1 in cardiomyocytes. Atf3 regulates expression of downstream genes and is implicated in negative feedback regulation of other immediate early genes. To identify Atf3-regulated genes, we knocked down Atf3 expression in cardiomyocytes exposed to endothelin-1 and used microarrays to interrogate the transcriptomic effects. Of upregulated mRNAs, expression of 23 (including Egr1, Ptgs2) was enhanced and expression of 25 was inhibited by Atf3 knockdown. Using quantitative PCR, we determined that knockdown of Atf3 had little effect on upregulation of Egr1 mRNA over 30 min, but abolished the subsequent decline, causing sustained Egr1 mRNA expression and enhanced protein expression. This resulted from direct binding of Atf3 to the Egr1 promoter. Mathematical modelling established that Atf3 can suffice to suppress Egr1 expression. Given the widespread co-regulation of Atf3 with Egr1, we suggest that the Atf3-Egr1 negative feedback loop is of general significance. Loss of Atf3 caused abnormal cardiomyocyte growth, presumably resulting from dysregulation of target genes. Our data therefore identify Atf3 as a nexus in cardiomyocyte hypertrophy required to facilitate the full and proper growth response.